Raman reaction device for in-situ quantitative monitoring of microorganism gas production process

Abstract

The invention belongs to the technical field of microorganism gas production monitoring, and particularly relates to a Raman reaction device for in-situ quantitative monitoring of a microorganism gas production process, the top of a main cabin body is in threaded connection with a cabin cover B, a butyl rubber plug is arranged in a top opening of the main cabin body, and an immersed Raman probe penetrates through the cabin cover B and the butyl rubber plug to be inserted into the main cabin body; bidirectional sealing is realized through a butyl rubber plug; the lower end of the immersed Raman probe is wrapped by a high-temperature-resistant tissue culture sealing film; gas / liquid channels are formed in the left side and the right side of the main cabin body, a T-shaped butyl rubber plug and a butyl rubber gasket which achieve double sealing are arranged in the gas / liquid channel in each side, and the gas / liquid channel in each side is in threaded connection with a cabin cover A; the high-precision digital pressure gauge is connected with a sterile syringe needle, and the high-precision digital pressure gauge penetrates through a cabin cover A, a T-shaped butyl rubber plug and a butyl rubber gasket in the gas / liquid channel on one side through the sterile syringe needle and enters the main cabin body. The gas tightness is good, and interference of steam water and liquefied water on Raman signals is effectively prevented.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial gas production monitoring, in particular to a Raman reaction device for in-situ quantitative monitoring of a microbial gas production process. Background Art

[0002] In the research and industrial application of anaerobic microorganisms, the gas production behavior of microorganisms is an important indicator to measure their metabolic characteristics. Traditional gas production monitoring methods mainly rely on offline analytical methods such as gas chromatography. However, these methods usually require the separation of the culture device from the analytical instrument and measure the gas composition and concentration by sampling. There are generally limitations such as cumbersome operation, easy introduction of external air pollution, and inability to achieve real-time monitoring. It is difficult to meet the current needs of dynamic research on complex microbial metabolic activities. In order to meet the needs of real-time and in-situ monitoring, with the development of spectral analysis technology, some improved reaction chambers have also begun to combine spectral technology. Laser Raman spectroscopy has received widespread attention in the field of microbial gas metabolism research due to its advantages of rapid, non-destructive, and in-situ detection. However, the existing Raman detection device is relatively simple in design and single in function. It is mostly used for the analysis of liquid or solid samples. The Raman reaction system for gas still has significant deficiencies in sealing performance, detection sensitivity, and high temperature and high pressure resistance.

[0003] In addition, the anaerobic microbial culture process usually requires sterilization, inoculation and culture operations to be completed under strict anaerobic conditions, which places high demands on the airtightness of the reaction device, the high temperature resistance of the materials and the ease of operation. Traditional anaerobic culture bottles are prone to sample leakage during gas sampling, and there is a large error in the absolute concentration quantification of the gas in the reaction system. This limitation significantly affects the accurate assessment of the microbial gas production process. Therefore, the development of a device that can meet the needs of continuous culture of anaerobic microorganisms and real-time gas component monitoring, especially while ensuring sealing, high temperature resistance and ease of operation, to achieve absolute concentration quantification of gas production components, is a technical problem that urgently needs to be solved in this field.

[0004] Specifically, the existing Raman reaction devices used for microbial gas production research have the following main disadvantages:

[0005] (1) Insufficient quantitative capability: Although laser Raman spectroscopy has the advantage of non-destructive testing, existing devices lack the absolute quantitative capability of gas concentration when quantitatively analyzing mixed gas components, and can usually only perform semi-quantitative operations on gas component concentrations.

[0006] (2) Poor sealing performance: The sealing design of existing reaction chambers mostly adopts single-layer sealing, such as ordinary rubber gaskets or simple rubber plugs, which cannot withstand the airtightness requirements under high temperature and high pressure sterilization conditions. During frequent operations, the sealing parts are prone to wear, resulting in an increased risk of gas leakage during the experiment, thereby affecting the accuracy of the test results.

[0007] (3) The spectral signal-to-noise ratio is low: The laser Raman probe is usually connected to the reaction chamber in the form of an external window. The excitation light and the recovered light need to penetrate the window glass. Multiple optical reflections and transmission losses lead to a significant reduction in signal intensity.

[0008] (4) The inner surface of the window is easily contaminated by microbial attachments or liquefied water during the experiment, further reducing the quality of the Raman signal.

[0009] (5) Lack of multi-parameter joint measurement capability: Existing devices can usually only measure gas composition or pressure, and cannot simultaneously obtain multi-parameter data such as temperature, pressure, and gas concentration, making it difficult to fully characterize and optimize the reaction process. Summary of the invention

[0010] In order to solve the problem that the gas cannot be accurately qualitatively and quantitatively determined during the cultivation of gas-producing anaerobic microorganisms, and the concentration changes of gas components cannot be monitored in real time in situ, the purpose of the present invention is to provide a Raman reaction device for in situ quantitative monitoring of the gas production process of microorganisms. The Raman reaction device can be widely used in anaerobic microbial research, and can realize continuous monitoring of gas components and contents by combining Raman spectroscopy technology; and after obtaining environmental temperature and pressure data, the absolute molar concentration of each component is calculated using the ideal gas state equation.

[0011] The objective of the present invention is achieved through the following technical solutions:

[0012] The present invention comprises a main cabin, an immersion Raman probe and a high-precision digital pressure gauge, wherein a hatch cover B is threadedly connected to the top of the main cabin, a butyl rubber plug is arranged in the top opening of the main cabin, the immersion Raman probe passes through the hatch cover B and the butyl rubber plug and is inserted into the interior of the main cabin, and a bidirectional seal between the immersion Raman probe and the top opening of the main cabin is achieved through the butyl rubber plug; the lower end of the immersion Raman probe is located in the main cabin and is wrapped by a high-temperature resistant tissue culture sealing film, and the upper end of the immersion Raman probe is The end is located outside the main cabin body; gas / liquid channels are opened on the left and right sides of the main cabin body, and a T-shaped butyl rubber plug and a butyl rubber gasket for realizing double sealing are arranged in the gas / liquid channel on each side, and a hatch A is threadedly connected to the gas / liquid channel on each side; the high-precision digital pressure gauge is connected to a sterile syringe needle, which passes through the hatch A, the T-shaped butyl rubber plug and the butyl rubber gasket in the gas / liquid channel on one side and enters the main cabin body, thereby realizing real-time monitoring of the internal pressure of the main cabin body.

[0013] Wherein: a distance is left between the lower end of the high temperature resistant tissue culture sealing film and the lower end of the immersed Raman probe.

[0014] The high temperature resistant tissue culture sealing film is wrapped around the lower end of the immersed Raman probe, and the high temperature resistant tissue culture sealing film is fixed by the Raman probe O-ring in the form of the high temperature resistant tissue culture sealing film being sheathed around the lower end of the immersed Raman probe.

[0015] The gas / liquid channels opened on both sides are located on the left and right sides of the axial section of the main cabin and have a height difference. The high-precision digital pressure gauge enters the main cabin from the gas / liquid channel on the lower side through a sterile syringe needle.

[0016] The hatch cover A is provided with a plurality of sterile syringe needle passage holes.

[0017] The main cabin is made of high borosilicate glass.

[0018] The advantages and positive effects of the present invention are:

[0019] Aiming at the special needs of anaerobic microbial culture, the present invention solves the problem of reduced air tightness of traditional rubber plugs due to multiple needle punctures. The butyl rubber material used has excellent needle resistance, and the double-layer sealing design (T-type butyl rubber plug and butyl rubber gasket) further enhances the air tightness. After sterilization is completed, the butyl rubber gasket can be removed, and only the T-type butyl rubber plug can be used for inoculation operation, and then the butyl rubber gasket that has not been punctured can be reinstalled, thereby significantly reducing the loss of air tightness. The present invention also directly seals the submerged Raman probe inside the cabin, and wraps the lower end of the probe with a high-temperature resistant tissue culture sealing film, which effectively prevents steam water and liquefied water from interfering with the Raman signal, greatly improving the signal-to-noise ratio of the spectral data. At the same time, the real-time pressure data inside the main cabin is directly monitored by a high-precision digital pressure gauge, and the ideal gas state equation (PV=nRT, P is pressure (Pa), V is gas volume (m 3 ), T is temperature (K), n is the amount of gas substance (mol), and R is the molar gas constant J / (mol·K)) to achieve quantitative analysis of the absolute molar concentration of gas components. This design effectively makes up for the deficiency of traditional Raman spectroscopy technology in only semi-quantitative analysis of mixed gas components. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is a structural front view of the present invention;

[0021] Figure 2 It is a left side view of the structure of the present invention;

[0022] Figure 3It is a top view of the structure of the present invention;

[0023] Among them: 1 is the main cabin, 2 is the hatch A, 3 is the T-shaped butyl rubber plug, 4 is the butyl rubber gasket, 5 is the left gas / liquid channel, 6 is the hatch B, 7 is the immersed Raman probe, 8 is the butyl rubber plug, 9 is the high temperature resistant tissue culture sealing film, 10 is the Raman probe O-ring, 11 is the right gas / liquid channel, 12 is the sterile syringe needle, 13 is the high-precision digital pressure gauge, and 14 is the sterile syringe needle channel hole. DETAILED DESCRIPTION

[0024] The present invention will be further described below in conjunction with the accompanying drawings.

[0025] like Figures 1 to 3 As shown, the present invention comprises a main cabin 1, an immersion Raman probe 7 and a high-precision digital pressure gauge 13, wherein a hatch cover B6 is threadedly connected to the top of the main cabin 1, a butyl rubber plug 8 with a hollow structure is arranged in the top opening of the main cabin 1, the immersion Raman probe 7 passes through the hatch cover B6 and the butyl rubber plug 8 and is inserted into the interior of the main cabin 1, and a two-way seal between the immersion Raman probe 7 and the top opening of the main cabin 1 is achieved through the butyl rubber plug 8; the lower end of the immersion Raman probe 7 is located in the main cabin 1 and is wrapped by a high-temperature resistant tissue culture sealing film 9, and the upper end of the immersion Raman probe 7 is located at the main cabin 1 Outside; gas / liquid channels are provided on both sides of the main cabin 1, and a T-shaped butyl rubber plug 3 and a butyl rubber gasket 4 for realizing double sealing are provided in each gas / liquid channel, and a hatch A2 is threadedly connected to each gas / liquid channel; a high-precision digital pressure gauge 13 is connected to a sterile syringe needle 12, and the sterile syringe needle 12 passes through the hatch A2, the T-shaped butyl rubber plug 3 and the butyl rubber gasket 4 in the gas / liquid channel on one side and enters the main cabin 1, thereby realizing real-time monitoring of the internal pressure of the main cabin 1, and combining Raman spectral data and ambient temperature to realize absolute quantitative analysis of gas components.

[0026] The material of the main cabin 1 of this embodiment is high borosilicate glass, which has excellent high temperature and high pressure resistance and can withstand high temperature (such as 121°C) and high pressure (such as 101.5KPa) sterilization process under closed conditions. The internal volume of the main cabin 1 can be flexibly adjusted according to the scale requirements of microbial cultivation. In addition, the transparent property of glass allows the growth status to be observed in real time during the microbial cultivation process.

[0027] The gas / liquid channels opened on both sides of this embodiment are the left gas / liquid channel 5 and the right gas / liquid channel 11, respectively. The left gas / liquid channel 5 and the right gas / liquid channel 11 are located on the left and right sides of the axial section of the main cabin 1, and have a height difference. The left gas / liquid channel 5 is opened at a position close to the top opening of the main cabin 1, and the right gas / liquid channel 11 is opened at a position close to the bottom of the main cabin 1. The high-precision digital pressure gauge 13 enters the main cabin 1 through the sterile syringe needle 12 from the right gas / liquid channel 11 with a lower height. The left gas / liquid channel 5 and the right gas / liquid channel 11 both use T-shaped butyl rubber plugs 3 and butyl rubber gaskets 4 to achieve double-layer sealing, which effectively reduces the problem of decreased air tightness caused by multiple operations; then a complete sealing structure is formed with the main cabin 1 through the hatch A2. This design can ensure that the air tightness of the Raman reaction device is maintained under high temperature and high pressure sterilization conditions.

[0028] The high temperature resistant tissue culture sealing film 9 of this embodiment is wrapped around the lower end of the immersion Raman probe 7, and the high temperature resistant tissue culture sealing film 9 is fixed by the Raman probe O-ring 10, which is covered by the high temperature resistant tissue culture sealing film 9 on the lower end of the immersion Raman probe 7. The high temperature resistant tissue culture sealing film 9 wraps the immersion Raman probe 7 and extends to the lower end of the immersion Raman probe 7, that is, there is a gap between the lower end of the high temperature resistant tissue culture sealing film 9 and the lower end of the immersion Raman probe 7, so as to effectively prevent the steam water in the sterilization process and the liquefied water in the culture process from infecting the sapphire window at the lower end of the immersion Raman probe 7, while reducing the risk of contamination and significantly improving the collection quality of the Raman signal. The high temperature resistant tissue culture sealing film 9 of this embodiment is made of polypropylene, which can withstand a high temperature of 132°C.

[0029] The hatch cover A2 of this embodiment is provided with a plurality of sterile syringe needle passage holes 14 .

[0030] The measuring range of the high-precision digital pressure gauge 13 in this embodiment is 0-1.0 MPa, and the accuracy is 0.0001 MPa.

[0031] The hatch cover A2 in this embodiment is made of PP (polypropylene) and has a maximum temperature resistance of 145°C.

[0032] The hatch cover B6 in this embodiment is made of PBT (polybutylene terephthalate) material with a maximum temperature resistance of 180°C.

[0033] The working principle of the present invention is:

[0034] First, use the butyl rubber gasket 4, the T-shaped butyl rubber plug 3 and the hatch A2 to seal the right gas / liquid channel 11, and then inject an appropriate amount of liquid microbial culture medium. Inject the culture medium according to the scale of culture required for the experiment. In this embodiment, the internal space of the main cabin 1 is 300mL, and 0 to 100mL of liquid culture medium can be injected. If the internal space of the main cabin 1 is larger, the amount of culture medium that can be injected can also be increased accordingly.

[0035] Subsequently, the high temperature resistant tissue culture sealing film 9 and the Raman probe O-ring 10 are connected to the submerged Raman probe 7 and sealed to the main cabin 1 through the hatch B6. Next, the main cabin 1 is placed in an anaerobic glove box and vacuumed to remove the internal air (the air is discharged from the left gas / liquid channel 5). After the vacuuming is completed, fill with specific gas according to the needs of microbial culture, and seal the left gas / liquid channel 5 with a butyl rubber gasket 4, a T-shaped butyl rubber plug 3 and a hatch A2. According to the needs of the cultured microorganisms, the specific gas and proportion used for internal evacuation are adjusted accordingly; the specific gas in this embodiment is a mixed gas of N2, CO2 and H2, and the mixed volume ratio is 8:1:1.

[0036] Since the filling gas combinations that can be set in the anaerobic glove box are limited, the present invention also supports operation outside the anaerobic glove box:

[0037] First, seal the submerged Raman probe 7 and the right gas / liquid channel 11 according to the above steps, and inject a set amount of liquid microbial culture medium into the main cabin 1 from the left gas / liquid channel 5. The amount of liquid microbial culture medium injected should be enough to tilt the main cabin 1 to the left, so that the right gas / liquid channel 11 is completely exposed, and the liquid microbial culture medium does not flow out from the left gas / liquid channel 5. Next, unscrew the hatch A2 on the right gas / liquid channel 11, remove the butyl rubber gasket 4 and T-shaped butyl rubber plug 3 in the right gas / liquid channel 11, fill the required mixed gas from the right gas / liquid channel 11, and discharge the air from the left gas / liquid channel 5. After emptying, seal the left gas / liquid channel 5 and the right gas / liquid channel 11 in turn. After completing the emptying and gas filling, place the cabin in a sterilizing pot to sterilize the Raman reaction device and the liquid microbial culture medium. After sterilization is completed, wait for the liquid microbial culture medium to cool to room temperature, unscrew the hatch 2 and butyl rubber gasket 4 of the right gas / liquid channel 11, and use a disposable sterile syringe to inject the microorganisms and substrates into the liquid culture medium of the main cabin 1 through the T-shaped butyl rubber plug 3 to complete the inoculation. The substrate refers to the substance that is acted upon by the microorganism during the reaction or metabolism process. For example, during the cultivation process, methyl-type methanogenic archaea use methanol as a substrate for growth and development.

[0038] Subsequently, the right gas / liquid channel 11 is resealed. When the experiment is running, the excitation light irradiates the sample in the main cabin 1 through the immersed Raman probe 7, and the Raman signal is transmitted to the spectrometer for analysis through the probe. The pressure data is recorded in real time by a high-precision digital pressure gauge 13, and the temperature data depends on the environment in which the Raman reaction device is located, which can be placed outdoors or in a constant temperature cabinet under constant temperature conditions. The main cabin 1 is made of high borosilicate glass, which has excellent temperature and pressure resistance and can withstand high temperature and high pressure sterilization under closed conditions. Hatch cover A2 and hatch cover B6 are made of high temperature resistant material and can withstand temperatures of up to 145°C. T-type butyl rubber stopper 3, butyl rubber stopper 8 and other components (such as high temperature resistant tissue culture sealing film 9 and Raman probe O-ring 10) also have good temperature resistance and are suitable for the temperature and pressure conditions required for sterilization.

Claims

1. A Raman reaction device for in-situ quantitative monitoring of microbial gas production process, characterized in that: The invention comprises a main cabin (1), an immersion Raman probe (7) and a high-precision digital pressure gauge (13), wherein a hatch cover B (6) is threadedly connected to the top of the main cabin (1), a butyl rubber plug (8) is provided in the top opening of the main cabin (1), the immersion Raman probe (7) passes through the hatch cover B (6) and the butyl rubber plug (8) and is inserted into the interior of the main cabin (1), and a bidirectional seal is achieved between the immersion Raman probe (7) and the top opening of the main cabin (1) through the butyl rubber plug (8); the lower end of the immersion Raman probe (7) is located in the main cabin (1) and is wrapped by a high-temperature resistant tissue culture sealing film (9); the immersion Raman probe (7) is sealed with a sealing film (9) to prevent the immersion Raman probe from leaking. The upper end of (7) is located outside the main cabin (1); the left and right sides of the main cabin (1) are provided with gas / liquid channels, and each side of the gas / liquid channel is provided with a T-shaped butyl rubber plug (3) and a butyl rubber gasket (4) for achieving double sealing, and each side of the gas / liquid channel is threadedly connected to a hatch A (2); the high-precision digital pressure gauge (13) is connected to a sterile syringe needle (12), and the sterile syringe needle (12) passes through the hatch A (2), the T-shaped butyl rubber plug (3) and the butyl rubber gasket (4) in the gas / liquid channel on one side and enters the main cabin (1), thereby realizing real-time monitoring of the internal pressure of the main cabin (1).

2. The Raman reaction device for in-situ quantitative monitoring of microbial gas production process according to claim 1, characterized in that: A gap is left between the lower end of the high temperature resistant tissue culture sealing film (9) and the lower end of the immersed Raman probe (7).

3. The Raman reaction device for in-situ quantitative monitoring of microbial gas production process according to claim 1, characterized in that: The high temperature resistant tissue culture sealing film (9) is wrapped around the lower end of the immersed Raman probe (7), and the high temperature resistant tissue culture sealing film (9) is fixed by means of a Raman probe O-ring (10) in which the high temperature resistant tissue culture sealing film (9) is sheathed around the lower end of the immersed Raman probe (7).

4. The Raman reaction device for in-situ quantitative monitoring of microbial gas production process according to claim 1, characterized in that: The gas / liquid channels opened on both sides are located on the left and right sides of the axial section of the main cabin (1) and have a height difference. The high-precision digital pressure gauge (13) enters the main cabin (1) from the gas / liquid channel on the side with a lower height through a sterile syringe needle (12).

5. The Raman reaction device for in-situ quantitative monitoring of microbial gas production process according to claim 1, characterized in that: The hatch cover A (2) is provided with a plurality of sterile syringe needle passage holes (14).

6. The Raman reaction device for in-situ quantitative monitoring of microbial gas production process according to claim 1, characterized in that: The main cabin (1) is made of high borosilicate glass.

Images